Figure 1: Combinatorial strain engineering.

(a) A schematic illustrating the pathways rewired in Y. lipolytica’s metabolism to drastically increase lipogenesis capacity. (b) Nile red fluorescence analysis of the strains constructed in this study. Strain names include strain background (POlf, pex10, mfe1, pex10 mfe1), auxotrophies relieved (leucine⁺ or uracil⁺) and enzymes overexpressed. ‘Epi’ denotes episomal overexpressions. Lipogenesis is induced as pex10 deletion is coupled with leucine biosynthetic capacity and DGA1 overexpression—generally from front left to back right of the 3D contour heat map representation. Error bars represent the s.d of technical triplicates.(c) Fluorescence light microscopy images of six strains increasing in lipid content (white) from left (unmodified POlf) to right (pex10 mfe1 leucine⁺ DGA1). Scale bar, 20 μm. (d,e) Increases in lipid titre and lipid content realized by engineering POlf to create the pex10 mfe1 leucine⁺ uracil⁺ DGA1 strain and then optimizing fermentation conditions in a bioreactor. Error bars represent s.d. of technical triplicates. (f) Image of lysed pex10 mfe1 leucine⁺ uracil⁺ DGA1 (left) and POlf control (right) cells in 14ml culture tubes. Lipids remain floating after centrifugation.